Effect of Trichoderma viride on insoluble phosphorus absorption ability and growth of Melilotus officinalis

Phosphorus (Pi) deficiency is a major factor of limiting plant growth. Using Phosphate-solubilizing microorganism (PSM) in synergy with plant root system which supply soluble Pi to plants is an environmentally friendly and efficient way to utilize Pi. Trichoderma viride (T. viride) is a biocontrol agent which able to solubilize soil nutrients, but little is known about its Pi solubilizing properties. The study used T. viride to inoculate Melilotus officinalis (M. officinalis) under different Pi levels and in order to investigate the effect on Pi absorption and growth of seedlings. The results found that T. viride could not only solubilizate insoluble inorganic Pi but also mineralize insoluble organic Pi. In addition, the ability of mineralization to insoluble organic Pi is more stronger. Under different Pi levels, inoculation of T. viride showed that promoted the growth of aboveground parts of seedlings and regulated the morphology of roots, thus increasing the dry weight of seedlings. The effect of T. viride on seedling growth was also reflected the increasing of chlorophyll fluorescence parameters and photosynthetic pigment content. Moreover, compared to the uninoculated treatments, inoculation of T. viride also enhanced Pi content in seedlings. Thus, the T. viride was a beneficial fungus for synergistic the plant Pi uptake and growth.


Phosphorus (Pi) deficiency is a major factor of limiting plant growth. Using Phosphate-solubilizing microorganism (PSM) in synergy with plant root system which supply soluble Pi to plants is an environmentally friendly and efficient way to utilize Pi. Trichoderma viride (T. viride) is a biocontrol agent which able to solubilize soil nutrients, but little is known about its Pi solubilizing properties. The study used T. viride to inoculate Melilotus officinalis (M. officinalis) under different Pi levels and in order to investigate the effect on Pi absorption and growth of seedlings. The results found that T. viride could not only solubilizate insoluble inorganic Pi but also mineralize insoluble organic Pi.
In addition, the ability of mineralization to insoluble organic Pi is more stronger. Under different Pi levels, inoculation of T. viride showed that promoted the growth of aboveground parts of seedlings and regulated the morphology of roots, thus increasing the dry weight of seedlings. The effect of T. viride on seedling growth was also reflected the increasing of chlorophyll fluorescence parameters and photosynthetic pigment content. Moreover, compared to the uninoculated treatments, inoculation of T. viride also enhanced Pi content in seedlings. Thus, the T. viride was a beneficial fungus for synergistic the plant Pi uptake and growth.
Pi is an essential plant macronutrient required for growth and development 1 , which plays many key roles in plant life such as synthesis of nucleotides, composition of membranes, photosynthesis, respiration, carbohydrate transport, etc 2 . The main form of Pi used by plants is ortho-phosphate, but it is very easy to form insoluble compounds with Fe 3+ , Al 3+ , Ca 2+ and Mg 2+ in terrestrial ecosystems, so its low availability often limits the growth of plants 3 . Pi limitation is the main productivity limiting factor in agricultural 4,5 and grassland ecosystems 6 . Although the Pi deficiency in plants can be alleviated by applying soluble Pi fertilizer to plants, but the utilization rate of fertilizer is greatly reduced due to chemical fixation of Pi and agricultural runoff 7 . However, overuse of phosphate rock fertilizers can lead to eutrophication of water body and ultimately to environmental degradation. Some studies have shown that phosphate rock is a non-renewable resource, and phosphate reserves will be exhausted in 50-100 years 8,9 . Therefore, how plants use insoluble Pi in the soil is a hot issue at present.
Using PSMs as Pi biofertilizer has been reported to be an economical and environmentally friendly way to dissolve insoluble Pi in soils 10 . Phosphate-solubilizing microorganisms (PSMs) include phosphate-solubilizing bacteria (PSB) and phosphate-solubilizing fungi (PSF), normally PSF has higher phosphate-solubilizing activity 11 . In soil, PSB or PSF convert the insoluble Pi into soluble Pi for plant absorption and utilization by secreting organic acids and phosphatase 12 . PSMs have been shown to secrete phosphatases to hydrolyze organophosphorus in soil 13 . For example, the Bacillus subtilis strain KPS-11 could produce extracellular phytase to mineralize insoluble organophosphorus, which significantly promoted the absorption of insoluble organophosphorus in potatoes 14 . PSM is involved in solubilizing the insoluble inorganic Pi by producing organic acid e.g., gluconic, 2-ketogluconic acid, malic, lactic, acetic, citric, and succinic acid [15][16][17] . Some PSMs have two mechanisms at the same time, including Pi solubilization and Pi mineralization, but some PSMs have only one of them. For instance, Gomez-Ramirez et al. 18 found that the Bacillus spp. strain IBUN-02724 had two mechanisms of Pi solubilization/ mineralization at the same time, which not only was able to solubilize insoluble inorganic Pi from Ca 3 (PO 4 ) 2 and AlPO 4 , but also mineralize insoluble organic Pi from phytate.
Cultivation of seedlings. The seeds of M. officinalis were acquired from the Institute of Grassland Science of Northeast normal University, China. The seeds were soaked in 75% ethanol for 3 min, and followed by 2% NaOCl for 10 min with agitation. Then, sterilized seeds were germinated in plastic square pots ( O and 10 µM Fe-EDTA. The pH of the nutrient solution was 6.0. The 500 mL water was poured into the each pot every 5 days. Seedlings were grown for 30 days at 26 °C/22 °C in day/night temperature with a 16-h photoperiod, an irradiance of 480 µmol m −2 s −1 , and relative humidity 65-70%. The growth conditions during the experimental period were the same as that in different Pi stess treatment experiment.

Experimental design
The experiment was conducted using a two factor random block design with two factors, including different Pi forms and T. viride. The experiment designed eight treatments and each treatment had six replicates (Fig. 1b). The 1/2 strength Hoagland nutrient solution was used in this study containing two levels of Pi concentration, 500 μM KH 2 PO 4 (Pi) and 5 μM KH 2 PO 4 (1%Pi). The nutrient solution containing 5 μM KH 2 PO 4 was prepared by substituting KCl for KH 2 PO 4 so that the concentration of K in the nutrient solution was 0.5 mM for all treatments. Tricalcium phosphate (TCP) and egg yolk lecithin (Lecithin) was selected to be used as insoluble Pi. The applied treatments were Pi: irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 500 μM KH 2 PO 4 ; Pi + T: irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 500 μM KH 2 PO 4 and each seedling was inoculated with 5 mL of T. viride spores fluid; 1%Pi: irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 ; 1%Pi + T: irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 and each seedling was inoculated with 5 mL of T. viride spores fluid; 1%Pi + TCP: each pot containing TCP, and irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 ; 1%Pi + TCP + T: Each pot containing TCP, and irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 , and each seedling was inoculated with 5 mL of T. viride spores fluid; 1%Pi + Lecithin: Each pot containing Lecithin, and irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 ; 1%Pi + Lecithin + T: each pot containing Lecithin, and irrigating seedlings with 1/2 strength Hoagland nutrient solution containing 5 μM KH 2 PO 4 , and each seedling was inoculated with 5 mL of T. viride spores fluid. The Pi element applied in the treatment of Pi was 68 mg. The total amount of Pi in insoluble Pi treatments were the same as that in the treatment of Pi. Every 30-day-old seedlings were transplanted into plastic pots ( Fig. 1a). For this assay, the T. viride spores were inoculated into 100 mL of NBRIP liquid medium in 250 mL conical flasks and pH was adjusted to 7.0. The concentration of inoculated spores was 1 × 10 8 cfu·mL −1 and the proportion of inoculated spores was 2%. All treatments were incubated at 28 °C in the dark for 10 days. Then, the supernatants was tested for available Pi and mycelium was tested for total Pi using molybdenum blue method 36 . Culture filtrates was collected after centrifugation of 10-day-old and measured the pH value.
Determination of growth indices. The plant height and leaf area was measured using a precision ruler.
The formula for calculating leaf area was: leaf area (cm 2 ) = leaf length × leaf width × 0.75 37 . Stem diameter was measured using a vernier caliper. Shoots and roots were harvested from each pot and then the roots were washed by water to remove the vermiculite. The fresh shoots and roots were dried at 105 °C for 30 min and after that the temperature was reduced to 70 °C for 10 h. Using a balance with an accuracy of 1/10,000 to measure the dry weight of shoots and roots. The fresh washed roots were scanned using a desktop scanner (EPSON Perfection V 700 Photo; Epson, America, Inc., USA). The resulting image was processed using WinRHIZO image analysis system (Win RHIZO 2012 b; Regent, Canada) to determine the morphological characteristics of the roots. treatments were monitored using a MAXI-Imaging PAM M-Series (Heinz Walz GmbH, Effeltrich, Germany). All treatments were kept in a dark adaptation period of 20 min before measurements. The chlorophyll fluorescence parameters were analyzed, such as Maximum efficiency of PSII photochemistry (Fv/Fm), effective PSII quantum yield (ΦPSII or Y(II)), non-photochemical quenching (NPQ), photochemical quenching (qP) 35 . In order to determine the incidence of the irradiance level on the PSII fluorescence emissions, rapid light curves (RLCs) composed of 16  Determination of chlorophyll and carotenoid content. The top fresh leaves were cut off from each sample, and weighed to 0.5 g for extracting Chlorophyll (Chl). The leaves were immersed into 95% (v/v) ethanol until complete bleaching. The concentration was determined by measuring extract absorbances at 470 nm, 649 nm, and 665 nm in a spectrophotometer (Hitachi U-3000; Hitachi, Ltd., Chiyoda, Tokyo, Japan). The Chl a, Chl b, and carotenoid (Car) was calculated using the following formulas 38  Statement. Our research is in line with local and national guidelines.

Results
The ability to release insoluble Pi of T. viride and acidity in culture filtrates. The T. viride showed Pi solubilization activity in NBRIP medium along with a subsequent decrease in pH ( Growth indices. Pi content and T. viride was very important for seedling growth. The growth indices of the treatments with T. viride were higher than those without T. viride ( Table 2). The plant height in the 1%Pi + Lecithin + T treatment was 2.42% higher than the 1% Pi + TCP + T treatment. However, plant height of the 1%Pi treatment was the lowest among other treatments. Plant height in the 1%Pi + T treatment was on par with the 1%Pi treatment and the Pi + T treatment was pretty much the same as the Pi treatment. The stem diameter of the 1%Pi + Lecithin + T treatment was similar to the 1% Pi + TCP + T treatment, but were 10.14% and 22.76% higher than which treatments without T. viride, respectively. There was no significant difference between the Pi treatment and the Pi + T treatment in leaf area. The leaf area in the 1%Pi + Lecithin + T treatment was 5.67%  www.nature.com/scientificreports/ higer than the 1% Pi + TCP + T treatment. The shoot dry weight of the Pi + T treatment was the highest than the other treatments. The shoot dry weight of the 1%Pi + Lecithin + T treatment was similar to the 1% Pi + TCP + T treatment, but were 54.02% and 34.30% higher than which treatments without T. viride, respectively. The root dry weight in the Pi treatment and the Pi + T treatment were similar and higher than the other treatments. The root dry weight of the 1% Pi + Lecithin + T was 19.23% higher than the 1%Pi + TCP + T treatment. There was a significant positive correlation between parameters of aboveground growth and total Pi content (Fig. 5).
The root morphological parameters of inoculated T. viride were higher than those of uninoculated treatments ( Table 3). The root morphological parameters of the 1%Pi + Lecithin + T treatment were higher than the 1%Pi + Lecithin treatment. The root morphological parameters of the 1%Pi + TCP + T treatment were higher than the 1%Pi + TCP treatment. The root morphological parameters of the Pi + T treatment were higher than the Pi treatment, and as well as the 1%Pi + T treatment which were higher than the 1%Pi treatment. There was a significant positive correlation between parameters of root morphology and total Pi content (Fig. 5).

Parameters of Chlorophyll a fluorescence. Compared to the non-inoculated treatment, the T. viride
inoculation increased Fv/Fm, ϕPSII and qP, but decreased NPQ (Fig. 2). The results showed that the highest Fv/Fm was the Pi + T treatment, and the lowest was the 1%Pi treatment (Fig. 2a). In insoluble-Pi treatments which inoculated T. viride, Fv/Fm of the 1%Pi + Lecithin + T treatment was higher than the 1%Pi + TCP + T treatment. In low Pi treatments, Fv/Fm of the 1%Pi + T treatment was higher than the 1%Pi treatment. The results showed that the highest ϕPSII was the Pi + T treatment, and the lowest was the 1%Pi treatment (Fig. 2b). In insoluble-Pi treatments which inoculated T. viride, there was no significant difference of ϕPSII between the 1%Pi + Lecithin + T treatment and the 1%Pi + TCP + T treatment. In low Pi treatments, ϕPSII of the 1%Pi + T treatment was higher than the 1%Pi treatment. The results showed that the highest qP was the Pi + T treatment, and the lowest was the 1%Pi treatment (Fig. 2c). In insoluble Pi treatments which inoculated T. viride, qP of the 1%Pi + Lecithin + T treatment was higher than the 1%Pi + TCP + T treatment. In low Pi treatments, qP of the 1%Pi + T treatment was higher than the 1%Pi treatment. From the resaults, we found that the highest NPQ were the 1%Pi treatment and the 1%Pi + T treatment, the lowest were the Pi treatment and the Pi + T treatment (Fig. 2d). In insoluble-Pi treatments which inoculated T. viride, NPQ of the 1%Pi + Lecithin + T treatment was lower than the 1%Pi + Lecithin treatment and the 1%Pi + TCP + T treatment was lower than the 1%Pi + TCP treatment. However, there was no significant difference of NPQ between the 1%Pi + Lecithin + T treatment and the 1%Pi + TCP + T treatment and the same as the 1%Pi + Lecithin treatment and the 1%Pi + TCP treatment. In low Pi treatments, there was no significant difference of NPQ between the 1%Pi treatment and the 1%Pi + T Table 2. Plant height and stem diameter and leaf area and shoot/root dry weight under different treatments. Values are the means ± S.E. (n = 6) based on analyses by one-way ANOVAs followed by S-N-K test. Different letters indicate significant difference (P < 0.05).

Treatment
Plant height (cm) Stem diameter (mm) Leaf area (cm 2 ) Shoot dry weight (g) Root dry weight (g) www.nature.com/scientificreports/ treatment. There was a significant positive correlation between parameters of chlorophyll a fluorescence and shoot total Pi content (Fig. 5).
We also analyzed the light-response curves of the Y (II), ETR, qP, and NPQ of PSII under the condition of the T. viride inoculation or non-inoculation in deffrent Pi supply (Fig. 3). Y(II) reduction of the inoculated T. viride treatment was more slowly than uninoculated T. viride treatment under light intensity (PAR < 500 µmol·m −2 ·s −1 ) (Fig. 3a). When light intensities > 500 µmol·m −2 ·s −1 , the production of light quantum production was inhibited. ETR increasing of the inoculated T. viride treatment was more faster than uninoculated T. viride treatment under a certain range of light intensity (PAR < 500 µmol·m −2 ·s −1 ), subsequently reached plateaued (Fig. 3b). qP of the T. viride inoculation treatment decreased slowly compared with the T. viride uninoculation treatment under light intensity (PAR < 500 µmol·m −2 ·s −1 ) (Fig. 3c). NPQ of the T. viride uninoculation treatment rose sharply relative to the T. viride inoculation treatment under light intensity (PAR < 500 µmol·m −2 ·s −1 ) (Fig. 3d). Excess excitation energy was dissipated as heat energy under the condition of low Pi and insoluble Pi. Therefore, inoculation with T. viride alleviated PSII impairment to some extent.
The content of chlorophyll, carotenoid and Chl a/Chl b ratio. The content of chlorophyll a, chlorophyll b and carotenoid content in the treatment with T. viride was significantly higher than that for the treatment without T. viride. There were no significant differences in Chl a/Chl b ratios among all treatments ( Table 4). The chlorophyll a content, chlorophyll b content, carotenoid content and Chl a/Chl b ratio in Pi + T was the highest than other treatments. In the Pi + T treatment, the chlorophyll a content was 14.69% higher than the Pi, but there was no significant difference in chlorophyll b content, carotenoid content, Chl a/Chl b ratio between the Pi + T treatment and the Pi treatment. The chlorophyll a content of the 1%Pi + Lecithin + T treatment was 41.35% higher than the 1%Pi + Lecithin treatment and the chlorophyll b content of the 1%Pi + Lecithin + T treatment was 11.94% higher than the 1%Pi + Lecithin treatment. However, there was no significant difference in carotenoid content, Chl a/Chl b ratio between the 1%Pi + Lecithin + T treatment and the 1%Pi + Lecithin treatment. The chlorophyll a content of the 1%Pi + TCP + T treatment was 27.62% higher than the 1%Pi + TCP treatment and the chlorophyll b content of the 1%Pi + TCP + T treatment was 13.85% higher than the 1%Pi + TCP www.nature.com/scientificreports/ treatment. However, there was no significant difference in carotenoid content, Chl a/Chl b ratio between the 1%Pi + Lecithin + T treatment and the 1%Pi + Lecithin treatment. There was no significant difference in Photosynthetic pigment parameters between the 1%Pi + T treatment and the 1%Pi treatment. photosynthetic pigment was positively correlated with aboveground growth Indices, chlorophyll a fluorescence except NPQ, and the correlation between Chl a/Chl b ratio and other parameters was not obvious (Fig. 5).    The content of Pi in shoot and root. Compared to the non-inoculated treatment, the T. viride inoculation increased the content of Pi in shoot and root. But there was no difference between the Pi treatment and the the Pi + T treatment, and which was no difference between the 1%Pi treatment and the the 1%Pi + T treatment (Fig. 4). On the other hand, plants grown with T. viride under insoluble Pi conditions were more easier to shift Pi from below-ground to above-ground tissues than the teatments without T. viride. In the 1%Pi + TCP + T treatment, the shoot Pi content was 65.46% higher than the root Pi content. In the 1%Pi + Lecithin + T treatment, the shoot Pi content was 114.43% higher than the root Pi content. The shoot Pi content was positively correlated with parameters of aboveground growth, root morphology, photosynthetic pigment, parameters of chlorophyll a fluorescence except NPQ (Fig. 5). The root Pi content was positively correlated with root dry weight, root morphology, but negatively correlated with NPQ (Fig. 5).

Discussion
In recent years, there has been an growing interest in PSM. The use of PSM is an effective way to dissolve insoluble Pi in the soil and thus supply it to plants for uptake. At the same time, it had a beneficial effect on the growth and development of plants. In agricultural production, the application of PSM to the soil could reduce the use of chemical fertilizer and gradually replace the dominant position of chemical fertilizer 39,40 . Therefore, the present study evaluated the role of T. viride in Pi solubilization and in promoting the growth of M. officinalis.
The results showed that the T. viride could dissolve TCP and lecithin and reduce the pH value in the culture medium (Table 1). This might be because the PSM can dissolve insoluble Pi by releasing protons and producing organic acids to reduce the surrounding pH 41,42 . However, dissolving Pi is not a simple phenomenon, and each kind of PSM can use a variety of mechanisms to dissolve insoluble Pi. Therefore, it is necessary to further study the Pi solubilization mechanism of T. viride.
Inoculation of PSM has been reported to not only dissolve insoluble Pi in soil, but also promote plant growth 43 . Our results showed that the growth indices of M. officinalis inoculated with T. viride were higher than the treatments without T. viride when insoluble Pi was used as Pi source ( Table 2). The choices of Pi source and the addition of T. viride had a very strong effect on the dry weight of M. officinalis. This finding is consistent with previous work by Abdenaceur et al., who found that Trichoderma spp. secrete plant growth-promoting hormones, such as IAA 44 . Root is an important organ for plants to absorb nutrients, which can sense the changes of nutrient concentration in soil, thus influencing changes in the root morphology of plants 45 . A study revealed that mung bean inoculated with PSB (Pseudomonas spp.) could increase root length and dry weight 46 . Our study also confirmed that the root morphological parameters of the treatment inoculated with T. viride were better than those without inoculation (Table 3). However, due to the presence of T. viride, M. officinalis changes its resource allocation strategy and is able to allocate more resources to above-ground growth and facilitate Pi uptake by the plant, allowing plants to have lower investment into below-ground biomass, and higher benefit for above-ground biomass (Table 3 and Fig. 4). Chlorophyll fluorescence parameters can characterize photosynthetic ability and energy conversion efficiency 47 . A study reported that under salt stress, inoculation of AMF or PSF increased the nutrient absorption of beach plums, and improved the parameters of chlorophyll fluorescence, such as Fv/ Fm, qP and ϕPSII values, but NPQ values remained unchanged or decreased compared with the control 48 . Our results showed that the values of Fv/Fm, qP and ϕPSII of M. officinalis inoculated with T. viride under insoluble Pi stress were higher than those of the treatments without T. viride, while the values of NPQ were lower than the treatments without T. viride (Fig. 2). Moreover, the Y (II) and qP light-response curves of M. officinalis inoculated with T. viride decreased slowly compared with that without inoculation (Fig. 3). The ETR light-response curves of M. officinalis inoculated with T. viride increased faster than that without inoculation. The NPQ light-response curves of M. officinalis inoculated with T. viride rise more slowly than that without inoculation. Our study suggested that M. officinalis inoculated with T. viride could increase the efficiency of excitation energy capture by leaf chloroplasts and increase the photochemical capacity of PSII. The content of plant photosynthetic pigment shows the degree of plant stress and can be used as an indicator to evaluate the physiological status of plants 49 . Liu et al. 50 found that the chlorophyll content of alfalfa inoculated with PSB was higher than that without inoculation. Similar to this study, the treatment inoculated with T. viride had higher Chlorophyll content and Carotenoid content and the Chl a/Chl b ratios than the uninoculated treatment (Table 4).
Qi et al. 51 found that AMF were able to promote Solidago canadensis absorb more Pi in insoluble Pi conditions. A study reported that when phosphate rock was used as Pi source, Trichoderma spp. could dissolve phosphate rock and enhance the Pi content of chickpea shoots and roots, such as T. viride, T. virens, T. virens 34 . Our results also proved this point, the content of Pi in shoots and roots of M. officinalis inoculated with T. viride was higher than that wuthout inoculation (Fig. 4). T. viride and Pi source are the main factors, which affecting the Pi uptake and biomass of M. officinalis. Moreover, when insoluble Pi was used as Pi source, the Pi content of M. officinalis inoculated with T. viride was more distributed to the stem and supplied to the aboveground part of the plant. However, the M. officinalis without T. viride had more Pi content in the root under the condition of insoluble Pi. This may be due to the fact that the root system is the main organ for plants to absorb nutrients and plants do not need to expend more resources to invest in root growth under the condition of sufficient Pi.

Conclusions
This study provides new evidence that T. viride can dissolve insoluble inorganic phosphorus and insoluble organic Pi. In addition to providing soluble phosphorus, T. viride also promotes plant growth of M. officinalis by improving root morphology and regulating plant photosynthesis. This research lays a foundation for recommending T. viride as a biological fertilizer and reduces environmental pollution caused by chemical fertilizer.